Myeloid cell leukemia-1 expression in cancers of the oral cavity: a scoping review

Background B cell lymphoma-2 (Bcl-2) family members play important roles in cell survival as well as cell death. The role of myeloid cell leukemia-1 (Mcl-1), an important member of the Bcl-2 family, is well established in hematopoietic malignancies. However, the association between Mcl-1 and oral cavity, cancers is not clearly defined. Methods A scoping review was conducted until June 30, 2021, using four major databases, PubMed, Scopus, Web of Science, and Embase. Medical subject headings keywords for Mcl-1, along with its other identifiers, and head and neck cancers (only oral cavity tumors) were used to evaluate the expression, function, molecular association, and therapeutic approach of Mcl-1 in oral cavity cancers and precancers. Findings Mcl-1 expression was associated with the progression of oral cavity cancers. The molecular mechanism and pathways of Mcl-1 in oral cavity cancers established via experimental results have been highlighted in this review. Moreover, the various synthetic and naturally derived therapeutic agents targeting Mcl-1 have been documented. Novelty/Improvement Based on our present review, Mcl-1 appears to be an effective anticancer target that can be used in the therapeutic management of oral cancers.

mitochondrial effects [9]. Just as anti-apoptotic Bcl-2 family members antagonize pro-apoptotic BH3-only proteins to inhibit the essential apoptosis effectors Bak/BAX [10], Mcl-1 exerts its anti-apoptotic function by sequestering the pro-apoptotic proteins Bak/ BAX [11]. Mcl-1 is regulated via modifications at the transcriptional, post-transcriptional, translational, or post-translational levels, and the functional activity and stability of Mcl-1 is determined by its post-translational modifications [12][13][14]. Notably, alternative splicing can specifically affect Mcl-1 function by yielding a longer isoform, which is anti-apoptotic, or a shorter isoform, which is pro-apoptotic [13].
Mcl-1 overexpression has been associated with poor outcomes and therapeutic responses in hematologic malignancies [15] and breast [16,17], lung [18], and gastric cancers [19]. Its overexpression in different cancers, particularly in leukemia, has resulted in an increased focus on the therapeutic targeting of this protein [20] leading to the development and identification of various synthetically produced, naturally occurring, or synthetically derived natural analogous compounds targeting Mcl-1 [21][22][23][24]. In addition to single compounds, combination therapies that target Mcl-1 reportedly show promising effects [24]. On the basis of the information currently available, we hypothesize that Mcl-1 can be a promising target for anticancer therapy.
The aim of the current review was to evaluate the expression, regulation, function, associated features, and potential therapeutic agents of Mcl-1 in oral cancers.

Methods
A previously established method was used to conduct a scoping review by applying the Preferred Reporting Items for Systematic Reviews and Meta-Analyses for Scoping Reviews guidelines.

Search strategy
A literature search was conducted using the PubMed, Scopus, Embase, and Web of Science databases as well as a gray literature search using Research Gate and Google Scholar until June 30, 2021. Medical subject headings (MeSH) terms were used to explore Mcl-1 along with other aliases, such as oral cancer, SGT, precancerous lesion, head and neck SCCs; other tumors were not included for this review. Only the studies published in English were evaluated, and duplicated records, posters, and abstracts were excluded (Fig. 1).

Eligibility criteria
The articles were reviewed by two authors of this study (SJC and NS) for eligibility and included after evaluation using the SPIDER criteria (Table 1).

Mcl-1 expression in clinical oral cancer samples
OPMLs have a high likelihood to progress to cancer, and the identification of oncogenic proteins that aid in the progression to oral cancer can be extremely helpful for better therapeutic planning. Several authors have verified that Mcl-1 is overexpressed in OPMLs. Ribeiro et al. [25] observed gains in Mcl-1 in two patients with leukoplakia and erythroleukoplakia. Similarly, Mallick et al. [26] reported the upregulation of Mcl-1 in malignant and premalignant tissues in vivo, interestingly indicating that the expression of Mcl-1 in homogeneous leukoplakia tended to be higher than in non-homogeneous leukoplakia. Our group also previously showed that Mcl-1 is overexpressed in oral lichen planus compared with the normal oral mucosa [27]. Sulkshane et al. reported that Mcl-1 was upregulated in OPMLs and demonstrated a positive correlation between Mcl-1 and USP9X in leukoplakia [28]. Moreover, Yu et al. found that an increase in the Bak/Mcl-1 ratio had favorable therapeutic outcomes after on photodynamic therapy for oral verrucous hyperplasia and leukoplakia [29]. These results indicate the essential role of Mcl-1 in the malignant transformation of OPMLs.
Mcl-1 overexpression is well documented in various solid and hematological tumors, including oral cancer, and has been demonstrated as genetic amplifications [25] and in mRNA [26,30,31] and protein [26,28,32,33] levels. According to a study by Nagata et al., strong Mcl-1 expression was observed in tongue SCC (SCCKN and SAS) cell lines compared with fibroblasts from normal lips [32]. The results of a study by Shin et al. [33] were valuable in terms of Mcl-1 expression through analysis of normal oral mucosa, human OSCC tissues (n = 14 and 25, respectively) and various OSCC cell lines (HSC2, HSC3, HSC4, HN22, OSC-20, Ca9.22, and SAS). In addition, Sulkshane et al. [28] confirmed the strong expression of Mcl-1 in other OSCC cell lines (AW8507, AW13516, and SCC029B). SGTs form a heterogeneous group of tumors that can be aggressive in nature; their gene expression patterns are similar to those of OPMLs and OSCC. The ubiquitous overexpression of Mcl-1 was reported in various types of malignant parotid gland tumors; the highest expression was observed in SCC of the parotid gland [34]. Although an isolated finding, Mcl-1 amplification was observed in high-grade stage III adenoid cystic carcinoma [35]. Determining the associations between Mcl-1 and the various categories and stages of oral   [28,30] and lymph node metastasis [30] are limited. Mcl-1 overexpression has been reported more in recurrent tumors than in primary tumors [28]. In addition, increased Mcl-1 expression has been associated with reduced overall survival [28,30], disease-free survival, and survival time [31,36]. Various histopathological indicators have been used to predict the progression of OSCC. Interestingly, increased Mcl-1 expression was associated with well-differentiated tumors [26,32]. Mcl-1 plays an important role in keratinocyte differentiation as it helps to maintain mitochondrial function [37]. These findings indicate a complex interaction, wherein histological function is maintained despite the poor clinicopathological stages. Taken together, the consistent findings of Mcl-1 overexpression in cancers indicates its association with carcinogenesis, and it is suggested that Mcl-1 has a significant impact on the development and progression of oral cancer. The associations between Mcl-1 and the different features of OPMLs, OSCC, and SGTs are summarized in Fig. 2; Table 2.  [32,38,39]. The activity of Mcl-1 in oral cancer is found to be regulated by paracrine signaling mechanisms, physical forces, or intracellular regulatory mechanisms [28,39,40]. The Mcl-1 mRNA expression was upregulated by STAT3 activation and stabilized by Akt-mediated GSK3β inactivation in chemotherapy-resistant OSCC [38]. The tumorigenesis regulating gene MYB is capable of upregulating Mcl-1 in adenoid cystic carcinoma cell lines [41]. FBW7 stabilizes Mcl-1 and promotes Mcl-1 addiction in oral cancer [42]. USP9X modulates the stability of Mcl-1 and prevents its degradation by deubiquitinating the protein [28]. Hyperosmotic stress has been shown to counteract Mcl-1 in head and neck SCC [39]. The upregulation of Noxa acts as a link between the osmotic pressure in the tumor environment and mitochondrial priming, thereby counteracting the anti-apoptotic properties of Mcl-1 in head and neck SCC. LncRNA FGD-AS1 inhibited the proliferation and migration/invasion of oral cancer, acting as a sponge for miR-153-3p and miR-153-3p to inhibit Mcl-1 expression [43]. Furthermore, the noncoding RNA HOXA10 AS was found to increase Mcl-1 mRNA levels [44]. Mcl-1 function can be also regulated through alternative splicing; a study demonstrated that Mcl-1 L transcripts were highly expressed compared with those of Mcl-1 S and Mcl-1ES in oral cells, thus indicating the predominance of the anti-apoptotic isoform [26,30]. This variation in the isoform has a significant impact on Mcl-1 function and even on its clinical presentation [30,45]. The effects of Mcl-1 on different oncogenic cascades have been evaluated in interference studies. Mcl-1 siRNA inhibited cell growth and induced apoptosis by inhibiting the FAK-MAPK pathway in OSCC [32]. Mithramycin inhibits Mcl-1 and RNAi regulates Bax to induce apoptosis in oral cancer cell lines [33]. These results suggest that Mcl-1 is affected and regulated by a variety of protein kinases, transcription factors, miRNA, etc. The molecular interactions and associations of Mcl-1 in oral cancers are summarized in Table 3, whereas and the protein-protein interactions (PPIs) between the identified biomarkers are presented in Fig. 3.

Therapeutic strategy targeting Mcl-1
Various compounds that can result in apoptosis can reduce the expression level of Mcl-1 by inhibiting its translation or increasing its rate of degradation. These compounds have been found to have an effect on the levels of Mcl-1 when used alone or in combination with other agents. Therefore, the key factors that inhibit Mcl-1 can be used as potential treatment strategies in the treatment of oral cancer.

Synthetic compounds
Several direct and indirect approaches to inhibit the activity of Mcl-1 have been used. Although small molecule inhibitors that directly target Mcl-1 by interrupting the PPIs have been developed, no drugs that can directly target this protein have been used in the treatment of oral cancer to date. Alternatively, some synthetic or natural compounds were found to target Mcl-1 indirectly as a part of their mechanism of action. A Bcl-2 inhibitor, obatoclax, was found to induce apoptosis in head and neck SCC in an Mcl-1-dependent manner [46]. ABT-737 repressed cellular Mcl-1 by upregulating Noxa [47]. TW-37 was reported to sensitize cryptotanshinone-mediated apoptosis in OSCC cells by suppressing STAT3-Mcl-1 signaling [48]. Furthermore, the proteasome inhibitor MG132 induced the accumulation of Bik, which can activate Bak sequestered by Mcl-1, to sensitize the TRAIL-mediated apoptosis [49]. Several kinase inhibitors have been shown to downregulate Mcl-1 in oral cancer; e.g., the aurora-A kinase inhibitor, alisertib, degraded Mcl-1 in HPV E7-expressing head and neck SCC cells [50]. Similarly, the multikinase inhibitor sorafenib induced apoptosis in mucoepidermod carcinoma cells through the STAT3/Mcl-1/t-Bid signaling Fig. 3 STRING protein-protein interaction (PPI) analyses. PPI network connectivity for proteins identified following the review. Nodes represent the proteins required for interaction. Edges represent the associations between the proteins. The STRING web resource (http:// www. strin gdb. org) was used in the prediction of the PPI (Protein-Protein Interaction) network whereby an interaction score of > 0.900 denoted a significant interactive relationship pathway [51]. EGFR inhibitors induced apoptosis in head and neck SCC by downregulating Mcl-1 expression [52,53]. Mithramycin A reduced the expression of Mcl-1 in oral cancer cells, leading to an increase in Bax protein, followed by its translocation into the mitochondria and oligomerization [33]. An HDAC inhibitor, panobinostat, suppressed Sp1 and downregulated Mcl-1 levels [54]. An inhibitor of the splicing factor 3B1, meayamycin B, reportedly to inhibited SF3B, leading to a reduction in the anti-apoptotic Mcl-1 L isoform and the generation of the pro-apoptotic Mcl1-S by switching the splicing pattern of the Mcl-1 pre-mRNA [55]. YM155 inhibited Mcl-1 through lysosomal-dependent degradation to induce apoptosis in head and neck SCC cell lines [56]. Aspirin downregulated the Mcl-1 protein, followed by a significant reduction in ERK-1/2 and Akt phosphorylation and significant increase in IκB-α phosphorylation, thus resulting in the activation of NF-κB [57]. The immunosuppressant FTY720 downregulated Akt/NF-κB signaling through a Mcl-1-dependent mechanism [58]. Propofol induced apoptosis via a significant reduction in Mcl-1 and an increase in phospho-Mcl-1 (Ser 159) thereby indicating its effect on the stability of Mcl-1 protein [59]. Biochemical synthetic products such as glucosamine hydrochloride and the anti-malaria semisynthetic dihydroartemisinin demonstrated a reduction in Mcl-1 in OSCC cell lines [60][61][62][63]. Several combination treatments affected the function of Mcl-1;e.g., a combination of fenretinide and ABT263 induced Mcl-1 degradation [64]. Co-treatment with C6 ceramide significantly augmented PKC412-induced lethality by downregulating Mcl-1 in head and neck cell lines and animal models [65]. These results suggest that synthetic compounds targeting Mcl-1 is a promising therapeutic strategy for the treatment of oral cavity cancers. The combination of thioridazine and carboplatin induced apoptosis by downregulating c-FLIP and Mcl-1 [66], indicating that Mcl-1 can be used as a molecular target of combination therapy in oral cancer. Clinical studies on Mcl-1 inhibitors are under way, and anticancer effects have been identified in several cancers other than those of the oral cavity [31]. Venetoclax and others drugs are under clinical trials for the treatment of acute myeloid leukemia and other hematological malignancies [24]. Table 4 summarizes various synthetic agents used to target Mcl-1.

Natural compounds
Many natural compounds are known to affect STAT3, which is known as one of the major upstream molecules of Mcl-1 in oral cancers [67]. Epigallocatechin gallate abrogated the interleukin-6-induced phosphorylation of STAT3 and downregulated its target gene products [68].
Licochalcone C inhibited the JAK2/STAT3 pathway, and downregulated Bcl-2 and Mcl-1 [69]. Nitidine chloride decreased the Mcl-1 protein by inhibiting the STAT3 pathway [70]. Additionally, bitter melon extract inhibited the c-Met signaling pathway and reduced the downstream signaling molecules such as phospho-STAT3 (Tyr705) and Mcl-1 [71]. These findings suggest that the STAT3/Mcl-1 signaling axis is a promising molecular mechanism that can be used in the treatment of oral cancers.
Various phytochemicals may mimic the effects of BH-3 proteins. Guggulsterone phytosterol targets 14-3-3 zeta to initiate apoptosis through the intrinsic mitochondrial pathway by the dephosphorylation of p-Bad and suppression of the expression level of Mcl-1 in OSCC cells [72]. Furano-1,2-naphthoquinone upregulated Bax and Bad and downregulated Mcl-1 in Ca9.22 cells [73]. Convallaria keiskei reduced the expression level of Mcl-1, leading to a truncated Bid-induced mitochondrial apoptosis in salivary gland cancer cell lines [74]. Lycorine hydrochloride induced the mitochondria-mediated apoptosis pathway through the downregulation of Mcl-1 [75]. Treatment with Juniperus squamata induced a mitotic catastrophe, leading to apoptosis via Mcl-1 reduction in OSCC cell lines [76].
Extracts from various plants were found to target Sp1, which combines with a specific DNA sequence and is overexpressed in many cancers [77]. Sp1, a transcription factor that binds to the Mcl-1 promoter region [78], has already been tested and found to play important physiological roles, such as in apoptosis, by targeting Mcl-1 in cancer [54,79]. Honokiol inhibited Sp1 and reduced Mcl-1 and survivin leading to the induction of apoptosis in OSCC cells [80]. Manumycin A inhibited Mcl-1 by downregulating Sp1 [81]. Sanguisorba officinalis [82] and C. officinale Makino, C. bursapastoris [83], and Dianthus chinensis and Acalypha australis [84] were found to reduce Mcl-1 via Sp1 and induce apoptosis in oral cancer cell lines.
ROS production results in a reduction in the mitochondrial transmembrane potential which leads to mitochondria-dependent apoptosis in human cancer cells [85]. ROS has been implicated in the activation of various cellular signaling pathways and transcription factors [86]. Phenethyl isothiocyanate induced G2/M cell cycle arrest and apoptosis by inducing ROS production and reducing Mcl-1 expression [87]. Benzyl isothiocyanate led to a reduction in Mcl-1 followed by the development of mitochondria-mediated apoptosis in oral cancer [88]. Cardiac glycosides induced apoptosis by lowering Mcl-1 levels in OSCC cell lines [89]. Wogonin was noted to selectively kill cisplatin-resistant head and neck SCC cells by targeting Nrf2, which was then accompanied by the        Bitter melon Momordica charantia BME treatment led to inhibition in cellular proliferation. The treatment led to the inhibition and downregulation of c-met and its downstream targets, such as phospho-STAT3 (Tyr705) and Mcl-1 (long anti-apoptotic form). Additionally, a reduction in c-myc was also observed.
Cal27 (tongue), JHU-22 (Larynx), JHU-29(tongue) [71]  downregulation of Mcl-1 [90]. Cyclocommunol downregulated the phosphorylation/expression of Akt/mTOR and Mcl-1 leading to the generation of ROS [91]. Taken together, the most commonly observed mechanism of action of these natural compounds in the regulation of Mcl-1 was through the inhibition of STAT3 or Sp1. Table 5 presents an overview of the effects of the natural compounds on Mcl-1.

Conclusions
In this paper, we attempted to review the expression, function, molecular mechanism and pathway, and therapeutic approach of Mcl-1 in oral cavity cancers. Mcl-1 is frequently amplified and upregulated in cancerous lesions of oral cavity and affects the clinical progression and survival of patients with oral cancer. Various transcription factors and protein kinases affect Mcl-1 activity, which further facilitates cancer progression. These findings indicate its significant role in oral carcinogenesis. This review also successfully summarized the agents, both synthetic and natural, that have an inhibitory effect on Mcl-1 in oral cancer. To the best of our knowledge, this review is the first specific summary suggesting that Mcl-1 is a promising molecular target for the treatment of oral cancer. Although the development of direct Mcl-1 inhibitors remains challenging, this review will help researchers and clinicians to identify the avenues that can be investigated to provide better disease prediction and therapeutic planning of oral cancers expressing Mcl-1 in the future.